This invention relates to a protein with an activity of the glucanosyltransferase type, and more especially a xcex2-(1-3)-glucanosyltransferase activity.
This invention also relates to oligonucleotides coding for this protein having an enzymatic activity.
It also relates to molecules having an effect on the activity of this enzyme.
Opportunistic fungal infections due to Candida, Aspergillus, Cryptococcus and Pneumocystis are responsible for the increase in morbidity and mortality among patients suffering from AIDS and other patients with clinically compromised immunity. In addition, the yeast Candida and the dermatophytes today remain a major medical problem amongst patients with adequate immunity, Despite the increase in the number of infections due to pathogenic and opportunistic fungi, therapy against mycoses has not improved in recent years. Two families of drugs are used: the azoles and Amphotericin B. These drugs have some disadvantages since treatment based on Amphotericin B is associated with nephrotoxicity and that based on azole is more fungistatic than fungicidal.
Fungi are microorganisms of the eukaryotic type which share the majority of their biochemical pathways with their hosts, with one important exception: the biosynthesis of the cell wall. The cell wall is a rigid envelope which protects the cell against the environment and mechanical stresses, but is also a dynamic structure which is involved in the transport of ions and macromolecules and in the localization of enzymes involved in fungal growth. In consequence, disorganization of the organization of the cell wall should be detrimental to fungi.
The skeleton of the fungal cell wall is mainly composed of polymers of the polysaccharide type (xcex2(1-3) glucans, mannans, chitin) which are not found in humans. For this reason, the biosynthesis of the cell wall has been a target for research into new antifungal drugs. The penicillins and cephalosporins, which are both inhibitors of the bacterial cell wall, and potential antibiotics lend support to this hypothesis. Moreover, many molecules which inhibit the development of the fungal cell wall have antifungal properties (Debono and Gordee, 1994, Annu. Rev. Microbiol, 48, 471-497). Among these are:
1) The families of the echinocandin lipopeptides and the palulacandin glycopeptides which are non-competitive inhibitors of the glucan synthetase complex.
2) The polyoxins and nikkomycins which are analogs of UDP-GlcNac and potential competitive inhibitors of chitin synthetase, and
3) The pradimycins binding mannan and the benanomycins.
The synthesis of xcex2(1-3) glucan and chitin is under the control of enzyme complexes (glucan synthetase and chitin synthetase) which are localized in the plasma membrane. Once the polymers have been released into the periplasmic space, cross-links are created between the polymers and it is these which are responsible for the rigidity of the cell wall. The proteins and genes of the glucan and chitin synthetases are beginning to be fairly well understood.
However, the inhibition of the glucan and chitin synthetases by a molecule requires three steps: its transfer across the cell wall, crossing of the plasma membrane and transfer inside the cell to the target, each step representing a potential barrier for the enzymatic inhibitor from being an effective antifungal drug, or a potential source of resistant strains against the drug.
The transferases which are responsible for creating the covalent bonds between the different polymers of the wall have been very little studied up till now.
These enzymes represent a better target than the chitin and glucan synthetase complexes since they are more easily accessible for a putative antifungal drug.
Nuoffer et al. (1991, Mol. Cell. Bio., 11, 27-37) have described a glycoprotein, named Gas1p, exposed on the surface of the yeast Saccharomyces cerevisiae. The genes coding for this protein have been cloned. The function of the Gas1p protein is not essential for the viability of the cell, and has not been determined.
Saporito-Irwing et al. (1995, Mol. Cell. Biol., 15, 601-613) have isolated a gene originating from the yeast Candida albicans, designated PHR1. The amino acid sequence determined for this protein PHR1 was 56% identical to that of the protein Gas1.
The gene was regulated in response to the pH of the culture medium. As for the protein gas1p, no function has been determined.
It clearly emerges from this analysis of the prior art that there has been a problem in obtaining molecules with effective antifungal activity.
The inventors have solved this problem.
They have shown that the introduction of mutations into a glucanosyltransferase originating from Aspergillus fumigatus interferes with the development of this micro-organism.
They have also determined the sequences of several of these enzymes.
The present invention thus relates to a first protein with an activity of the xcex2-(1-3)-glucanosyltransferase type characterized in that it has at least 50%, preferably 60%, and even more preferably 85% homology with proteins having the sequences, or a part of the sequences SEQ ID No 2 or SEQ ID No 3 as follows:
SEQ ID NO:2:
Met Lys Ala Ser Ala Val Thr Ala Ala Leu Ala Val Gly Ala Ser Thr Val Leu Ala Ala Pro Ser Ile Lys Ala Arg Asp Asp Val Thr Pro Ile Thr Val Lys Gly Asn Ala Phe Phe Lys Gly Asp Glu Arg Phe Tyr Ile Arg Gly Val Asp Tyr Gln Pro Gly Gly Ser Ser Asp Leu Ala Asp Pro Ile Ala Asp Ala Asp Gly Cys Lys Arg Asp Ile Ala Lys Phe Lys Glu Leu Gly Leu Asn Thr Ile Arg Val Tyr Ser Val Asp Asn Ser Lys Asn His Asp Glu Cys Met Asn Thr Leu Ala Asp Ala Gly Ile Tyr Leu Val Leu Asp Val Asn Thr Pro Lys Tyr Ser Ile Asn Arg Ala Lys Pro Lys Glu Ser Tyr Asn Asp Val Tyr Leu Gln Tyr Ile Phe Ala Thr Val Asp Ala Phe Ala Gly Tyr Lys Asn Thr Leu Ala Phe Phe Ser Gly Asn Glu Val Ile Asn Asp Gly Pro Ser Ser Ser Ala Ala Pro Tyr Val Lys Ala Val Thr Arg Asp Leu Arg Gln Tyr Ile Arg Ser Arg Lys Tyr Arg Glu Ile Pro Val Gly Tyr Ser Ala Ala Asp Ile Asp Thr Asn Arg Leu Gln Met Ala Gln Tyr Met Asn Cys Gly Ser Asp Asp Glu Arg Ser Asp Phe Phe Ala Phe Asn Asp Tyr Ser Trp Cys Asp Pro Ser Ser Phe Lys Thr Ser Gly Trp Asp Gln Lys Val Lys Asn Phe Thr Gly Tyr Gly Leu Pro Leu Phe Leu Ser Glu Tyr Gly Cys Asn Thr Asn Lys Arg Gin Phe Gln Glu Val Ser Scr Leu Tyr Ser Thr Asp Met Thr Gly Val Tyr Ser Gly Gly Leu Val Tyr Glu Tyr Ser Gln Glu Ala Ser Asn Tyr Gly Leu Val Glu Ile Ser Gly Asn Asn Val Lys Glu Leu Pro Asp Phe Asp Ala Leu Lys Thr Ala Phe Glu Lys Thr Ser Asn Pro Ser Gly Asp Gly Asn Tyr Asn Lys Thr Gly Gly Ala Asn Pro Cys Pro Ala Lys Asp Ala Pro Asn Trp Asp Val Asp Asn Asp Ala Leu Pro Ala Ile Pro Glu Pro Ala Lys Lys Tyr Met Thr Glu Gly Ala Gly Lys Gly Pro Gly Phe Ala Gly Pro Gly Ser Gln Asp Arg Gly Thr Gln Ser Thr Ala Thr Ala Glu Pro Gly Ser Gly Ser Ala Thr Gly Ser Ser Ser Ser Gly Thr Ser Thr Ser Ser Lys Gly Ala Ala Ala Gly Leu Thr Val Pro Ser Leu Thr Met Ala Pro Val Val Val Gly Ala Val Thr Leu Leu Ser Thr Val Phe Gly Ala Gly Leu Val Leu Leu
SEQ ID NO: 3: (BGT2)
Asp Asp Val Thr Pro Ile Thr Val Lys Gly Asn Ala Phe Phe Lys Gly Asp Glu Arg Phe Tyr Ile Arg Gly Val Asp Tyr Gln Pro Gly Gly Ser Ser Asp Leu Ala Asp Pro Ile Ala Asp Ala Asp Gly Cys Lys Arg Asp Ile Ala Lys Phe Lys Glu Leu Gly Leu Asn Thr Ile Arg Val Tyr Ser Val Asp Asn Ser Lys Asn His Asp Glu Cys Met Asn Thr Leu Ala Asp Ala Gly Ile Tyr Leu Val Leu Asp Val Asn Thr Pro Lys Tyr Ser Ile Asn Arg Ala Lys Pro Lys Glu Ser Tyr Asn Asp Val Tyr Leu Gln Tyr Ile Phe Ala Thr Val Asp Ala Phe Ala Gly Tyr Lys Asn Thr Leu Ala Phe Phe Ser Gly Asn Glu Val Ile Asn Asp Gly Pro Ser Ser Ser Ala Ala Pro Tyr Val Lys Ala Val Thr Arg Asp Leu Arg Gln Tyr Ile Arg Ser Arg Lys Tyr Arg Glu Ile Pro Val Gly Tyr Ser Ala Ala Asp Ile Asp Thr Asn Arg Leu Gin Met Ala Gln Tyr Met Asn Cys Gly Ser Asp Asp Glu Arg Ser Asp Phe Phe Ala Phe Asn Asp Tyr Ser Trp Cys Asp Pro Ser Ser Phe Lys Thr Ser Gly Trp Asp Gln Lys Val Lys Asn Phe Thr Gly Tyr Gly Leu Pro Leu Phe Leu Ser Glu Tyr Gly Cys Asn Thr Asn Lys Arg Gln Phe Gin Glu Val Ser Ser Leu Tyr Ser Thr Asp Met Thr Gly Val Tyr Ser Gly Gly Leu Val Tyr Glu Tyr Ser Gln Glu Ala Ser Asn Tyr Gly Leu Val Glu Ile Ser Gly Asn Asn Val Lys Glu Leu Pro Asp Phe Asp Ala Leu Lys Thr Ala Phe Glu Lys Thr Ser Asn Pro Ser Gly Asp Gly Asn Tyr Asn Lys Thr Gly Gly Ala Asn Pro Cys Pro Ala Lys Asp Ala Pro Asn Trp Asp Val Asp Asn Asp Ala Leu Pro Ala Ile Pro Glu Pro Ala Lys Lys Tyr Met Thr Glu Gly Ala Gly Lys Gly Pro Gly Phe Ala Gly Pro Gly Ser Gln Asp Arg Gly Thr Gln Ser Thr Ala Thr Ala Glu Pro Gly Ser Gly Ser Ala Thr Gly Ser Ser Ser Ser Gly Thr Ser Thr Ser Ser Lys Gly Ala Ala Ala Gly Leu Thr Val Pro Ser Leu Thr Met Ala Pro Val Val Val Gly Ala Val Thr Leu Leu Ser Thr Val Phe Gly Ala Gly Leu Val Leu Leu
This protein preferably has a molecular weight of about 44 kD, or of about 49 kD if it carries at least one residue of the N-glycosyl type.
The present invention also relates to proteins with xcex2-(1-3)-glucanosyltransferase activity characterized in that they have at least 50%, preferably 60% and even more preferably 85% homology with proteins having the sequences, or a part of the sequences SEQ ID No 10 or SEQ ID No 12 as follows:
SEQ ID No 10:
Gly Phe Phe Ala Gly Asn Glu Val Ile Asn Glu Gln Ser Val Lys Asn Val Pro Thr Tyr Val Arg Val Cys His Pro Ser Pro Gln Leu Thr Ile Ala Cys Pro Leu
SEQ ID No 12 (BGT4)
Gly Phe Phe Ala Gly Asn Glu Val Val Asn Gln Ala Asn Gln Ser Ala Gly Ala Ala Phe Val Lys Ala Ala Ala Arg Asp Met Lys Ala Tyr Ile Lys Thr Lys Gly Tyr Arg Gln Ser Leu Ala Ile Gly Tyr Ala Thr Thr Asp Asn Pro Glu Ile Arg Leu Pro Leu Ser Asp Tyr Leu Asn Cys Gly Asp Gln Ala Asp Ala Val Asp Phe Phe Gly Tyr Asn Ile Tyr Glu Trp Cys Gly Asp Gln Thr Phe Gln Thr Ser Gly Tyr Gln Asn Arg Thr Glu Glu Tyr Lys Asp Tyr Ser Ile Pro Ile Phe Ile Ser Glu Tyr Gly Cys Asn
The present invention also relates to fragments of these proteins.
Said invention is not limited to the proteins having the sequences SEQ ID No 2, SEQ ID No 3, SEQ ID No 10 or SEQ ID No 12 but extends to any protein having sequences similar to those having the sequences SEQ ID No 2, SEQ ID No 3, SEQ ID No 10 or SEQ ID No 12 and in particular having certain amino acid substitutions in which an amino acid is replaced by another amino acid having essentially the same physico-chemical properties. Lehninger""s biochemistry manual (Flammarion Medecine-Science, 1977, or one of its more recent editions) distinguishes four groups of amino acids, based on their physico-chemical behavior: those with a non-polar or hydrophobic side-chain, those with an uncharged polar side-chain, those with a negatively charged side-chain, and those with a positively charged side-chain.
The present invention also relates to nucleotide sequences coding for proteins, or protein fragments such as those described above, and more particularly DNA sequences (cDNA or genomic DNA) or RNA sequences.
Such a DNA sequence may be that having at least 50%, preferably 60% and even more preferably 85% homology with the genomic sequence SEQ ID No 1, or a part of the sequence SEQ ID No 1 as follows:
ATG AAG GCC TCT GCT GTT ACT GCC GCT CTC GCC GTC GGT GCT TCC ACC GTT CTG GCA GCC CCC TCC ATC AAG GCT CGT GAC GAC GTT ACT CCC ATC ACT GTC AAG GGC AAT GCC TTC TTC AAG GGC GAT GAG CGT TTC TAT ATT CGC GGT GTC GAC TAC CAG CCC GGT GGC TCC TCC GAC CTG GCT GAT CCC ATC GCT GAT GCC GAT GGT TGC AAG CGT GAC ATT GCC AAG TTC AAG GAG CTG GGC CTG AAC ACT ATC CGT GTC TAC TCG GTC GAC AAC TCC AAG AAC CAC GAT GAG TGT ATG AAT ACA CTG GCT GAT GCT GGC ATC TAT CTG GTG CTC GAT GTC AAC ACT CCC AAG TAC TCC ATC AAC CGC GCC AAG CCT AAG GAG TCG TAC AAC GAT GTC TAC CTC CAG TAT ATC TTC GCT ACC GTT GAT GCT TTC GCC GGT TAC AAG AAC ACC CTC GCT TTC TTC TCC GGC AAC GAG GTT ATC AAC GAT GGC CCT TCC TCC TCT GCT GCT CCC TAC GTC AAG GCC GTC ACT CGT GAT CTG CGT CAG TAC ATC CGT AGC CGC AAG TAC CGT GAG ATT CCT GTC GGC TAC TCG GCT GCC GAT ATC GAC ACC AAC CGT CTT CAG ATG GCC CAG TAT ATG AAC TGC GGT TCC GAC GAC GAG CGC AGT GAC TTC TTC GCT TTC AAC GAC TAC TCC TGG TGC GAT CCC TCC TCT TTC AAA ACC TCG GGC TGG GAT CAG AAG GTC AAG AAC TTC ACT GGC TAC GGT CTT CCT CTC TTC CTG TCC GAA TAC GGC TGC AAC ACC AAC AAG CGT CAA TTC CAA GAA GTC AGC TCT CTC TAC TCC ACG GAC ATG ACT GGT GTC TAC TCT GGT GGT CTC GTG TAC GAG TAC TCT CAG GAG GCC AGC AAC TAC GGT CTG GTG GAG ATT AGC GGC AAC AAT GTC AAG GAG CTC CCA GAC TTC GAC GCT CTG AAG ACC GCG TTC GAA AAG ACC TCC AAC CCC TCC GGC GAC GGC AAC TAC AAC AAG ACT GGT GGT GCC AAC CCT TGC CCC GCT AAG GAC GCT CCC AAC TGG GAC GTT GAC AAC GAT GCT CTT CCT GCC ATC CCC GAG CCC GCC AAG AAG TAC ATG ACT GAG GGT GCT GGC AAG GGC CCT GGT TTT GCC GGA CCT GGC AGC CAG GAC CGT GGT ACC CAG TCC ACT GCC ACT GCT GAG CCC GGA TCT GGC TCT GCC ACT GGA AGC AGC AGC AGC GGC ACC TCC ACC TCT TCC AAG GGC GCT GCA GCT GGC CTG ACT GTC CCT AGC CTG ACC ATG GCT CCC GTT GTC GTT GGT GCG GTT ACA CTC CTG TCC ACC GTC TTC GGC GCT GGC CTC GTC CTC TTG T GA
This sequence has been included in a 2.2 kb fragment, which has itself been included in the X bal site of the pUC19 vector (Maniatis et al., 1989, Cold Spring Harbor Laboratories Press). The strain E. coli DH5xcex1 carrying this modified vector was deposited in the Collection Nationale de Culture de Micro-Organismes at the Institut Pasteur (CNCM) on the Jul. 26, 1996 under the number I-1763.
Such a sequence may also be that having at least 50%, preferably 60% and even more preferably 85% homology with the complementary DNA sequence comprised in a 1.4 kb fragment, which has been included in the pCRII vector (In Vitrogen). This, carried by the E. coli DH5xcex1 strain, was deposited in the Collection Nationale de Culture de Micro-Organismes at the Institut Pasteur (CNCM) on the Jul. 26, 1996 under the number I-1762.
These two strains are objects of the present invention.
Nucleotide sequences according to the present invention may also be those having at least 50%, preferably 60%, and even more preferably 85% homology with one of the DNA sequences SEQ ID No 9 or SEQ ID No 11 as follows:
SEQ ID No 9:
GGCTTCTTCG CCGGCAACGA GGTTATCAAC GAGCAGAGTG TCAAGAACGT TCCCACTFAC GTCCGGGTAT GTCATCCATC CCCACAGCTT ACGATTGCCT GTCCACTGAC ACTCTCGTAG GCGACTCAGC GTGACATGAA GGACTACTAC GCAAAGAACC TTGACCGCAG CATTCCTGTT GGCTATTCTG CTGCCGATAT TCGTCCCATC CTCATGGCAC CCCTCAACTA CTTCATGTGC GCTGACGATG CTAATTCCCA ATCGGACTTC TTCGGCCTCA ACTCCTACTC GTGGTGCGGC AACTCGTCCT ACACCAAGAG TGGCTACGAT GTCCTCACCA AGGACTTTGC CGACGCCTCT ATCCCCGTCT TCATCTCCGA ATTCGGCTGC AACA
SEQ ID No 11
GGTTTCTTCG CCGGCAACGA GGTTGTGAAT CAGGCGAATC AGTCCGCCGG CGCTGCATTC GTCAAGGCCG CCGCGCGAGA CATGAAGGCC TACATCAAGA CCAAGGGATA CCGGCAATCG CTGGCAATTG GATACGCGAC CACTGACAAC CCGGAAATCC GACTCCCGCT GTCCGACTAC CTCAACTGCG GCGACCAGGC CGACGCGGTC GACTTCTTCG GCTACAACAT CTACGAATGG TGCGGTGACA AGACCTTCCA GACCTCGGGC TACCAGAACC GCACCGAGGA GTACAAGGAC TACTCCATCC CCATCTTCAT CTCCGAATAC GGCTGCAAC
These two sequences have been independently included in the pCRII vectors and introduced into the E. coli DH5xcex1 strain. These strains were deposited in the Collection Nationale de Culture de Micro-Organismes at the Institut Pasteur (CNCM) on the Aug. 22, 1997 under the numbers I-1914 and I-1913.
A further object of the present invention is a method for the detection of proteins with a strong homology with the sequence of the protein BGT2.
The present invention thus relates to a method for detecting a nucleotide sequence having at least 60% identity with the sequence SEQ ID No 1 in a biological sample containing nucleotide sequences, comprising the following steps:
a) placing the biological sample in contact with the nucleotide primers P3 and P4 having the sequences SEQ ID No 7 and SEQ ID No 8, respectively, as follows:
SEQ ID No 7:
GSYTTCTTCK CYGGCAACGA GGTT
SEQ ID No 8:
GTTGCAGCCG WATTCGGASA YGAA
xe2x80x83the nucleotide sequences contained in the sample having been if necessary put into a form enabling their hybridization under conditions enabling the hybridization of the primers with the nucleotide sequences.
b) amplification of the nucleotide sequences
c) revealing the amplification products, and
d) detection of the mutations by appropriate methods.
The proteins according to the invention may be obtained by purification of an autolysate of Aspergillus fumigatus. The protein may be purified by four steps of ion-exchange chromatography and one step of gel filtration.
Said proteins may also be obtained by genetic engineering methods. For example, the sequence SEQ ID No 1, if possible without its C-terminal part, may be cloned in an appropriate vector, and expressed in an expression system, such as the Pichia pastoris system, marketed by In Vitrogen.
In this system, the sequence of the gene coding for the protein is cloned in an expression vector, then linearized. Protoplasts originating from P. pastoris are transformed with the linearized vector.
The clones, in which a recombination is performed and which replaces the aoxl sequence by the sequence of the gene of the protein which it is desired to produce, are selected for their capacity to grow in a histidine-deficient medium. A person skilled in the art may refer to xe2x80x9cManual of methods for expression of recombinant proteins in Pichia pastorisxe2x80x9d, published by In Vitrogen.
The protein thus expressed, if possible secreted in the culture medium, is recovered by processes known to a person skilled in the art.
Such a protein may be used, in particular, for screening molecules to identify their antifungal activity.
Thus, another object of the present invention is a process for screening molecules to identify their antifungal activity comprising the following steps:
placing together the molecules to be screened and the protein or the protein fragment as described above, or coded by a sequence such as described above, and
determining the effect of the molecule on said protein.
The determination of the effect of the molecules on said protein may be accomplished by measuring the activity of the xcex2-(1-3) glucanosyltransferase (BGT2). Such activity may be determined by placing said protein in the presence of a substrate on which it has an effect, which may be composed of laminarioligosaccharides comprising at least 10 glucosyl radicals linked by xcex2-(1-3) bonds. When the protein is active, it cleaves a part of the molecule and binds the fragment obtained onto the non-reducing terminal of an uncleaved substrate molecule.
The product resulting from the activity of the protein is in the form of coupling products of two laminarioligosaccharides. This product may be detected by any process allowing the separation of oligosaccharides with different degrees of polymerization, in particular by chromatographic methods, such as high-pressure liquid chromatography (HPLC) or thin-layer chromatography (TLC). This latter method, although less precise than the first, is the easier to use.
For the use of these chromatographic methods, a person skilled in the art may consult the following manual: Carbohydrate analysis : a practical approach. Chaplin and Kennedy, 1986 IRC Press, Oxford.
This detection method enables determination as to whether the molecules detected have antifungal activity.
These molecules having antifungal activity show effects on the xcex2-(1-3)glucanosyltransferase activity of said proteins. These effects may be for example the inhibition of this activity.
The present invention also relates to molecules having an effect on said proteins, which may be detected by the process as described above, as well as the use of these molecules to prepare a drug, or for the treatment of diseases related to fungi, in vertebrates and plants.
One of the advantages of the use of these molecules lies in the low frequency of appearance of resistant strains of the fungi, in contrast to other known antifungal molecules.